DETAILED ACTION
Claims withdrawn in prior Office Action remain withdrawn as shown in PTO-892. Specifically, claims 4, 10-14 and 16-20 are withdrawn.
Prior objections and rejections not restated below are withdrawn.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-3 and 5-8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Iwasaki et al. (JP 2009065839 A) (JP839) further in view of Chen et al. (Rational Engineering of p-Hydroxybenzoate Hydroxylase to Enable Efficient Gallic Acid Synthesis via a Novel Artificial Biosynthetic Pathway, Biotechnol. Bioeng. 114, 2017, 2571-80) as evidenced by Entsch et al. (Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site directed mutants, J Biol Chem 266, 1991) 17341–17349).
A machine translation of JP839 is provided and cited herein.
JP 839, abstract, teaches:
To provide a new method for producing gallic acid. <P>SOLUTION:This method for producing the gallic acid is characterized by producing, accumulating and collecting the gallic acid from protocatechuic acid with a microorganism expressing a protein mentioned in the following (A) or (B). (A) The protein having an amino acid sequence represented by sequence numbers 16, 26, 30, 32 or 34, and (B) the protein having an amino acid sequence represented by the sequence numbers 16, 26, 32 or 34, wherein one or more amino acids are replaced, deleted, inserted or added.
“Regarding the method for producing gallic acid using an enzyme, there is no report on the production of gallic acid using a wild-type enzyme, but position 385 of the wild-type parahydroxybenzoic acid hydroxylase possessed by Pseudomonas aeruginosa PAO strain. It has been reported that gallic acid can be produced via protocatechuic acid starting from parahydroxybenzoic acid in a test tube by using a mutant enzyme (hereinafter abbreviated as PAO mutant enzyme) in which tyrosine is converted to phenylalanine.” JP839, page 1.
“An object of the present invention is to produce protocatechuic acid from an inexpensive raw material such as terephthalic acid, phthalic acid, isophthalic acid, or parahydroxybenzoic acid, and a protein having protocatechuic acid 5-position oxidizing activity (hereinafter referred to as protocatechuate 5-position oxidase). ) To provide a method for producing gallic acid at low cost by converting protocatechuic acid to gallic acid.” JP839, page 2.
“Database search of proteins having homology with the amino acid sequence of Pseudomonas arginosa PAO strain parahydroxybenzoate hydroxylase Pseudomonas arginosa PAO strain parahydroxybenzoate hydroxylase oxidizes parahydroxybenzoic acid. It is known that it possesses the activity of producing protocatechuic acid, but does not possess the activity of producing protocatechuic acid 5-position oxidation activity [Entsch, B,]. Among proteins having homology with the hydroxylase but not having a large homology value, Pseudomonas arginosa PAO strain has an activity different from that of parahydroxybenzoate hydroxylase, that is, protocatechuic acid 5-position oxidation activity. I decided to find out if I had the protein I had. Specifically, the protein having 75% or less homology with the hydroxylase is GenBank (hereinafter abbreviated as GB) of National Center for Biotechnology Information (hereinafter abbreviated as NCBI). The database was searched using the BLAST homology analysis method using the amino acid sequence of parahydroxybenzoic acid hydroxylase of Pseudomonas arginosa PAO strain shown in SEQ ID NO: 2 as a query sequence. As a result, 12 types of proteins shown in Table 1 were obtained. Abbreviations of each protein and Pseudomonas arginosa PAO. Table 1 shows the percent homology of the strains with parahydroxybenzoate hydroxylase.” JP839, Example 1.
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“10 types of 12 types of HFM enzymes (HFM5, HFM86, HFM145, HFM305, HFM339, HFM388
, HFM544, HFM545, HFM689 and HFM737) synthesized gallic acid when protocatechuic acid was used as a substrate.” JP839, page 13.
An alignment between recited SEQ ID NO: 2 and SEQ ID NO: 12 of JP839 is as follows:
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In summation of above, JP839 teaches that a wild-type parahydroxybenzoic acid hydroxylase from Pseudomonas aeruginosa PAO having SEQ ID NO: 2 does not have activity to form protocatechuic acid starting from parahydroxybenzoic acid as a substrate, but that a mutant of the same enzyme (Y385F) does have such activity. JP839 identifies various wild-type enzymes by sequence identity to SEQ ID NO: 2 of JP839 as reported in Table 1 wherein 10 of 12 identified enzymes have activity to produce gallic acid. It is noted that SEQ ID NO: 12 (HMF122) did not have activity for forming gallic acid, like the wild-type enzyme of SEQ ID NO: 2 of JP839.
JP839 teaches that various of the enzymes taught in Table 1 thereof can be mutated to have 385F (or corresponding substitution) in order to further benefit activity for production of gallic acid. “Construction of plasmids expressing mutant enzyme protein HFM145Y385F, mutant enzyme protein HFM305Y386F and mutant enzyme protein HFM6895Y384 The enzyme that has been proven to have the activity of generating gallic acid from protocatechuic acid is the 385th tyrosine of enzyme protein HFM300.” JP839, page 14; see also, JP839, claim 1. It is noted that the mentioned mutations is of the same corresponding position between the enzymes.
Since JP839 teaches that introduction of Y385F mutation as taught introduces activity for generating gallic acid from protocatechuic acid in hydroxylase enzymes from Pseudomonas aeruginosa PAO having SEQ ID NO: 2 of JP839 (wherein SEQ ID NO: 2 lacks such activity), at the time of filing an ordinarily skilled artisan would have been motivated to introduce such a Y385F mutation into any of the hydroxylase enzymes described in Table 1 of JP839, including SEQ ID NO: 12 of JP839, with a reasonable expectation of success in introducing activity for gallic acid production activity as taught to be desirable by JP839.
However, JP839 does not teach or suggest SEQ ID NO: 12 having a substitution of position 294 to alanine as recited in the claims.
Chen, abstract, teaches “structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) [i.e. 3,4-diydroxybenzoic acid] than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59mg/L [GA gallic acid] from 1000mg/L 4-hydroxybenzoic acid (4-HBA). “Remarkably, the Y385F/T294A mutant demonstrated enhanced catalytic activity toward 3,4-DHBA. It has a kcat value of 1.69+/00.18 s-1 toward 3,4-DHBA, which is a 4-fold increase when
compared with the reported Y385F mutant.
The p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa referenced by Chen is understood to the same enzyme as SEQ ID NO: 2 of JP839, which has the same alignment for numbering as SEQ ID NO: 12 of JP839 (and recited SEQ ID NO: 2) as shown in the alignment above. More specifically, the “Entsch et al., 1991” reference cited on page 2575 (left col.) of Chen is the same reference referred to in the first paragraph of Example 1 of JP839 such that the same enzyme is under discussion by both references. This reference is cited as an evidentiary reference.
JP839 does not teach or suggest SEQ ID NO: 12 having a substitution of position 294 to alanine as recited in the claims. However, as far as JP839, as discussed above, suggest making a Y385F mutation to SEQ ID NO: 12 of JP839 to increase ability to produce gallic acid, an ordinarily skilled artisan at the time of filing would have been further motivated to make additional mutations taught by the prior art to combined with Y385F including T294A as taught by Chen in order to increase activity for producing gallic acid. Upon making such modifications to SEQ ID NO: 12 of JP839, a p-hydroxybenzoate hydroxylase having over 99% identity to recited SEQ ID NO: 2 with substitution T294A is obtained.
Regarding recitation in the claims that an embodiment hydroxylase has 4-aminobenzoic acid hydroxylation activity, the specification directly evidences that a hydroxylase identical to SEQ ID NO: 2 (SEQ D NO: 12 of JP839) with substitution at positions 385 and 294 has 4-aminobenzoic acid hydroxylation activity. The cited prior art meets the features of claim 1 and the method features of “substituting an amino acid residue” as recited in claims 2 and 3 wherein any improvement in 4-aminobenzoic acid hydroxylation activity is inherent to making the substitution T294A.
Regarding claims 5-8, JP839, pages 14-15 explains that any mutated hydroxylase enzyme is produced by forming an encoding polynucleotide cloned into a vector and transformed in to a host cell (E. coli). As far as an ordinarily skilled artisan at the time of filing would have been motivated to produce a hydroxylase enzyme being SEQ ID NO: 12 of JP839 with substitionT294A/Y395F (as discussed above), the same artisan would have been motivated to produce the same using the same standard recombinant protein production techniques taught by JP839 in order to achieve the benefit of producing such a hydroxylase.
Allowable Subject Matter
Claims 9 and 15 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter:
Microbial production of 4-amino-3-hydroxybenzoic acid has been reported in the prior art, for example, Nadeau et al. (Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups, Appl. Environ. Microbiology 69, 2003, 2786-93). However, such conversion is by a mutase to convert “4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate.” Nadeau, abstract.
Regarding production of 4-amino-3-hydroxybenzoic acid from a 4-aminobenzoate by a hydroxylase, “The potential substrate [of P. fluorescnes p-hydroxybenzoate hydroxylase] p-aminobenzoate is very slowly hydroxylated by the wild-type enzyme because flavin reduction is rate-limiting.” Eschrich et al. (Role of Tyr201 and Tyr385 in substrate activation by p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, Eur. J. Biochem. 216, 1993, 137-46), page 141, left col. “The wild-type p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas fluorescens or Pseudomonas aeruginosa has high hydroxylase activity toward 4-hydroxybenzoic acid (4-HBA), but very weak or negligible activity toward 3,4-DHBA. In the previous studies, to approach this issue, the tyrosine at 385th position of PobA was mutated to phenylalanine to allow the enzyme to hydroxylate 3,4-DHBA into GA. However, with this mutation, the turnover rate of PobA toward the native substrate 4-HBA decreases to 2% of wild-type.” Chen (as cited above), page 2572, left col. Table 3 of Eschrich reports that the reduction rate of various hydroxylase mutants is significantly slower for 4-aminobenzoate as a substrate as compared to 4-hydroxybenzoate. As such, the cited art indicates 1) hydroxylases of the class consistent with the hydroxylases recited in the claims utilizes 4-hydroxybenzoate poorly, and 2) substitution of known amino acid residues for modifying hydroxylase activity have widely varying effects for different substrates. For these reasons, the teachings of the prior art of record are considered to insufficient to suggest transformation of the recited substituted hydroxylases specifically into a microorganism expressing 4-aminobenzoic acids, particularly since it is not predictable regarding whether such substituted hydroxylases will utilize 4-hydroxybenzoate even more poorly than unsubstituted hydroxylases in view of the prior art cited herein. Further, the allowed claims of U.S. 11,312,980 (not citable as prior art due to same inventorship) are referenced.
The possibility of a double patenting rejection over Ser No. 17/791,377 and 17/268,689 (U.S. 11,312,980) has been considered. However, the copending/issued claims do not recite any specific substitutions and there is no sufficient reason from the prior art why the 4-aminobenzoic acid hydroxylase recited in the copending/issued claims will benefit from substitution. That is, in view of the discussion above that substitution of active site residues in this class of enzyme can decrease activity to “native substrate 4-HBA decreases to 2% of wild-type,” there is no particular motivation that the same will improve activity on 4-aminobenozoic acid or other substrates sufficient to specifically warrant a non-statutory double patenting rejection.
Response to arguments
Applicant argues:
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As an initial matter it is noted that protocatechuic acid and 3,4-dihydroxybenzoic acid (3,4-DHBA) are synonyms for the same compound.
Chen is considered the closes prior art of record since the same is the only reference that teaches the substitution Y395F/T294, which is not taught by JP839. It does not appear to be disputed that hydroxylase enzymes with over 90% identity to SEQ ID NO: 2 are known in the prior art. Therefore, the point of novelty is the substitutions.
Chen, page 2572, left col., teaches the following:
The wild-type p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas fluorescens or Pseudomonas aeruginosa has high hydroxylase activity toward 4-hydroxybenzoic acid (4-HBA), but very weak or negligible activity toward 3,4-DHBA. In the previous studies, to approach this issue, the tyrosine at 385th position of PobA was mutated to phenylalanine to allow the enzyme to hydroxylate 3,4-DHBA into GA (Entsch et al., 1991). However, with this mutation, the turnover rate of PobA toward the native substrate 4-HBA decreases to 2% of wild-type. In addition, the Y385F mutant can only convert about half of 3,4-DHBA into GA from 0.5 mM 4-HBA by in vitro conversion.
PobA from P. aeruginosa is SEQ ID NO: 2 of JP839, which it the search sequence to identify the other hydroxylase enzymes discussed by JP839 including SEQ ID NO: 12 of JP839 that is over 99% identical to recited SEQ ID NO: 2.
SEQ ID NOS: 2 and 12 of JP839 share the property of having very week or negligible activity towards 3,4-DHBA to produce gallic acid. However, Chen provides a technical solution to this problem, which is introduction of two substitutions Y385F/T294A “which displayed much higher activity towards 3,4-dihydroxybenzymoic acid (3,4-DHBA) than the wild-type and any other reported mutants.” Chen, abstract. This is illustrated in Table III and Fig. 4 of Chen (reproduced below):
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Applicant argues a PHOSITA would focus only on a wild-type enzyme that already displays “strong gallic acid synthetic activity.” However, the authors of Chen are understood to be PHOSITAs and evidence the skill and motivations of a PHOSITA. The PHOSITAs of Chen, at a publication date several years after JP839, show that a PHOSITA at the time of filing would have been motivated to introduce substitutions Y385F/T294A into a P. aeruginosa p-hydroxybenzoate hydroxylase having very weak or negligible activity toward 3,4-DHBA, since the same is exactly what was done by Chen. As such, an ordinarily skilled artisan would have been motivate to introduce substitutions Y385F/T294 into other PobA enzymes having a relationship to P. aeruginosa p-hydroxybenzoate hydroxylase (SEQ ID NO: 2 of JP839) included those identified by JP839 (i.e. including SEQ ID NO: 12 of JP839) even if such wild-type enzyme ahs “very week or negligible activity toward 3,5-DHBA. It is understood at all times that the effect of introduction of substitutions is not absolutely predictable in the art. “Obviousness does not require absolute predictability, but at least some degree of predictability is required.” MPEP 2143.02(II). Here, the large degree of success for introducing hydroxylase activity toward 3,4-DHBA demonstrated by Chen provides the degree of predictability to motivate an ordinarily skilled artisan at time of filing to introduce the same substitutions into related PobA enzymes, including SEQ ID NO: 12 of JP839) to observe if the same increase in hydroxylase activity for 3,4-DHBA is observed.
Both JP839 and Chen recognize that introduction of Y385F only is a lacking technical solution, which partially motivates JP839 searching for enzymes having some endogenous 3,4-DHBA hydroxylase activity. Regardless, Chen explicitly teaches that superior 3,4-DHBA activity can be obtained by introducing substitutions Y385F/T294 into a p-hydroxybenzoate hydroxylase having very weak or negligible activity toward 3,4-DHBA. Further, JP839 has not teachings regarding introduction of substitutions Y385F/T294 and as such cannot teach away from introducing these substitutions into a p-hydroxybenzoate hydroxylase having very weak or negligible activity toward 3,4-DHBA as taught by Chen, since JP839 is completely silent regarding Y385F/T294 and therefore cannot teach away from a technical solution that is not discussed. Further, the publication date of Chen is several years after JP839 such that Chen represents the state of the art at time of filing. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues:
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Chen, page 2572, left col., teaches the following:
The wild-type p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas fluorescens or Pseudomonas aeruginosa has high hydroxylase activity toward 4-hydroxybenzoic acid (4-HBA), but very weak or negligible activity toward 3,4-DHBA. In the previous studies, to approach this issue, the tyrosine at 385th position of PobA was mutated to phenylalanine to allow the enzyme to hydroxylate 3,4-DHBA into GA (Entsch et al., 1991). However, with this mutation, the turnover rate of PobA toward the native substrate 4-HBA decreases to 2% of wild-type. In addition, the Y385F mutant can only convert about half of 3,4-DHBA into GA from 0.5 mM 4-HBA by in vitro conversion.
The examiner disagrees that the P. aeruginosa PobA enzyme of Chen characterized as “very week or negligible activity towards 3,4-DHBA” is “enhancing an enzyme that was already known to have some level of enzymatic activity.” Rather, this is a characterization that the enzyme is inactive with 3,4-DHBA for any practical purpose as taught by JP839. P. aeruginosa PobA enzyme is SEQ ID NO: 2 of JP839 that JP839 characterizes as inactive. “It [the P. aeruginosa hydroxylase] is known that it possesses the activity of producing protocatechuic acid, but does not possess the activity of producing protocatechuic acid 5-position oxidation activity [i.e. hydroxylation of 3,4-DHBA to gallic acid].” SEQ ID NO: 12 of JP839 is described to have the same property of activity with 3,4-DHBA that is so low as to be activity that is not possessed just as the P. aeruginosa PobA enzyme.
Applicant argues:
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The burden is on applicant to establish that results are unexpected and significant. MPEP 716.02(b). "[A]ppellants have the burden of explaining the data in any declaration they proffer as evidence of non-obviousness." Ex parte Ishizaka, 24 USPQ2d 1621, 1624 (Bd. Pat. App. & Inter. 1992); MPEP 716.02(b). “An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.” MPEP 216.02(e). "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602, 604 (CCPA 1967); MPEP 716.02(c)(II).
The closest prior art of record is Chen demonstrating a P. aeruginosa PobA enzyme with substitutions Y385F/T294; however, no comparison of the claimed subject matter to this closest prior art has been made. It is noted that claim 1 recites a composition of matter and the scope of the claim does not required the claimed hydroxylase enzymes to be utilized on an aminobenzoic acid substrate.
Conclusion
The grounds of rejection under 35 U.S.C. 103 are unmodified.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652